Stringent control during carbon starvation of marine Vibrio sp. strain S14: molecular cloning, nucleotide sequence, and deletion of the relA gene.

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain. The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E. coli RelA protein. The amino acid sequences are 64% identical, and they share some completely conserved regions. A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene. The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation. During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed. Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the [ATP]/[ADP] ratio. In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation. The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp. strain S14.     

Cephamycin C production is regulated by relA and rsh genes in Streptomyces clavuligerus ATCC27064

The effects of growth rate and nutrient uptake rate on the production of cephamycin C were determined in the parental strain, deltarelA mutant, and deltarsh null mutant of S. clavuligerus. Production of cephamycin C was inversely related to mycelium growth and the phosphate feed rate was more critical for the production of cephamycin C. On the contrary, the production of cephamycin C was completely abolished in the deltarelA mutant, but not in deltarsh mutant. The changes in the cephamycin C production by disruption of the relA and rsh genes are presumably associated with the consequent ability of the mutants to accumulate (p)ppGpp under nutrient starvation. Therefore, it is concluded that the stringent response of S. clavuligerus to starvation for nutrients is governed mainly by RelA rather than Rsh and that the response is more apparently regulated by the limitation of phosphate.     

Temperature-sensitive growth and decreased thermotolerance associated with relA mutations in Escherichia coli

The relA gene of Escherichia coli encodes guanosine 3',5'-bispyrophosphate (ppGpp) synthetase I, a ribosome-associated enzyme that is activated during amino acid starvation. The stringent response is thought to be mediated by ppGpp. Mutations in relA are known to result in pleiotropic phenotypes. We now report that three different relA mutant alleles, relA1, relA2, and relA251::kan, conferred temperature-sensitive phenotypes, as demonstrated by reduced plating efficiencies on nutrient agar (Difco) or on Davis minimal agar (Difco) at temperatures above 41 degrees C. The relA-mediated temperature sensitivity was osmoremedial and could be completely suppressed, for example, by the addition of NaCl to the medium at a concentration of 0.3 M. The temperature sensitivities of the relA mutants were associated with decreased thermotolerance; e.g., relA mutants lost viability at 42 degrees C, a temperature that is normally nonlethal. The spoT gene encodes a bifunctional enzyme possessing ppGpp synthetase and ppGpp pyrophosphohydrolase activities. The introduction of the spoT207::cat allele into a strain bearing the relA251::kan mutation completely abolished ppGpp synthesis. This ppGpp null mutant was even more temperature sensitive than the strain carrying the relA251::kan mutation alone. The relA-mediated thermosensitivity was suppressed by certain mutant alleles of rpoB (encoding the beta subunit of RNA polymerase) and spoT that have been previously reported to suppress other phenotypic characteristics conferred by relA mutations. Collectively, these results suggest that ppGpp may be required in some way for the expression of genes involved in thermotolerance.     

Global analysis of the carbon starvation response of a marine Vibrio species with disruptions in genes homologous to relA and spoT.

The stringent control response, which involves a rapid accumulation of ppGpp, is triggered if the marine Vibrio sp. strain S14 is subjected to carbon and energy starvation. By means of high-resolution two-dimensional gel electrophoresis analysis, we addressed the role of the major ppGpp-synthesizing enzyme (RelA) in the regulation of the carbon starvation response of Vibrio sp. strain S14. The finding that a large number of the carbon starvation-induced proteins were underexpressed in the Vibrio sp. S14 relA mutant strain after the onset of glucose starvation suggests that a rapid accumulation of ppGpp is required for induction of many of the carbon starvation-induced proteins. However, it was also found that a majority of the carbon starvation-induced proteins were significantly less induced if the stringent control response was provoked by amino acid starvation. We therefore also addressed the notion that a carbon starvation-specific signal transduction pathway, complementary to the stringent control, may exist in Vibrio sp. strain S14. It was found that a majority of the proteins that were underexpressed in the relA mutant strain were also underexpressed in the Vibrio sp. S14 spoT mutant strain (csrS1). Interestingly, a large proportion of these underexpressed proteins were found to belong to a group of proteins that are not, or significantly less, induced by starvation conditions that do not promote starvation survival. On the basis of these observations and the finding that the csrS1 strain survives poorly but accumulates ppGpp in a fashion similar to the wild type during carbon and energy source starvation, the gene product of the csrS gene is suggested to be responsible for the mediation of a signal which is complementary to ppGpp and essential for the successful development of the starvation- and stress-resistant cell. This conclusion was also supported by experiments in which changes in phenotypic characteristics known to be induced during carbon starvation were studied. The starvation induction of the high-affinity glucose uptake system was found to be dependent on the csrS gene but not relA, and the synthesis of carbon starvation-specific periplasmic space proteins was dependent, at different times of starvation, on both the relA and the csrS gene products.     

Accumulation of ppGpp in a relA mutant of Escherichia coli during amino acid starvation

In Escherichia coli, amino acid starvation triggers the rapid synthesis of two guanosine polyphosphates, pppGpp and ppGpp (the 3'-pyrophosphates of GTP and GDP, respectively). Determination of the turnover rate of the ppGpp pool indicated that during serine deprivation, as opposed to other amino acid starvations, the rate of ppGpp degradation is dramatically decreased. This results in a slow but significant accumulation of this regulatory nucleotide in a relA mutant during serine starvation. Similar ppGpp accumulation can be seen during serine starvation in different serine auxotrophic mutants carrying different relA alleles. On the other hand, no ppGpp accumulation is induced in various relaxed strains by serine hydroxamate treatment.     

The stringent response is required for amino acid and nitrate utilization, nod factor regulation, nodulation, and nitrogen fixation in Rhizobium etli

A Rhizobium etli Tn5 insertion mutant, LM01, was selected for its inability to use glutamine as the sole carbon and nitrogen source. The Tn5 insertion in LM01 was localized to the rsh gene, which encodes a member of the RelA/SpoT family of proteins. The LM01 mutant was affected in the ability to use amino acids and nitrate as nitrogen sources and was unable to accumulate (p)ppGpp when grown under carbon and nitrogen starvation, as opposed to the wild-type strain, which accumulated (p)ppGpp under these conditions. The R. etli rsh gene was found to restore (p)ppGpp accumulation to a DeltarelA DeltaspoT mutant of Escherichia coli. The R. etli Rsh protein consists of 744 amino acids, and the Tn5 insertion in LM01 results in the synthesis of a truncated protein of 329 amino acids; complementation experiments indicate that this truncated protein is still capable of (p)ppGpp hydrolysis. A second rsh mutant of R. etli, strain AC1, was constructed by inserting an Omega element at the beginning of the rsh gene, resulting in a null allele. Both AC1 and LM01 were affected in Nod factor production, which was constitutive in both strains, and in nodulation; nodules produced by the rsh mutants in Phaseolus vulgaris were smaller than those produced by the wild-type strain and did not fix nitrogen. In addition, electron microscopy revealed that the mutant bacteroids lacked poly-beta-hydroxybutyrate granules. These results indicate a central role for the stringent response in symbiosis.     

Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations

It was known previously that 1) the relA gene of Escherichia coli encodes an enzyme capable of guanosine 3',5'-bispyrophosphate (ppGpp) synthesis, 2) an uncharacterized source of ppGpp synthesis exists in relA null strains, and 3) cellular degradation of ppGpp is mainly due to a manganese-dependent ppGpp 3'-pyrophosphohydrolase encoded by the spoT gene. Here, the effects of spoT gene insertions and deletions are compared with analogous alterations in neighboring genes in the spo operon and found to be lethal in relA+ strains as well as slower growing in relAl backgrounds than delta relA hosts. Cells with null alleles in both the relA and spoT genes are found no longer to accumulate ppGpp after glucose exhaustion or after chelation of manganese ions by picolinic acid addition; the inability to form ppGpp is reversed by a minimal spoT gene on a multicopy plasmid. Strains apparently lacking ppGpp show a complex phenotype including auxotrophy for several amino acids and morphological alterations. We propose that the SpoT protein can either catalyze or control the alternative pathway of ppGpp synthesis in addition to its known role as a (p)ppGpp 3'-pyrophosphohydrolase. We favor the possibility that the SpoT protein is a bifunctional enzyme capable of catalyzing either ppGpp synthesis or degradation.     

Functional analysis of relA and rshA, two relA/spoT homologues of Streptomyces coelicolor A3(2)

Deletion of the (p)ppGpp synthetase gene, relA, of Streptomyces coelicolor A3(2) results in loss of production of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red) and delayed morphological differentiation when the mutant is grown under conditions of nitrogen limitation. To analyze the role of (p)ppGpp as an intracellular signaling molecule for the initiation of antibiotic production, several C-terminally deleted derivatives of S. coelicolor relA that could potentially function in the absence of ribosome activation were placed under the control of the thiostrepton-inducible tipA promoter. While 0.82- and 1.28-kb N-terminal segments failed to restore (p)ppGpp and antibiotic production upon induction in a relA null mutant, 1.46- and 2.07-kb segments did. Under conditions of phosphate limitation, deletion of relA had little or no effect on Act or Red synthesis, potentially reflecting an alternative mechanism for ppGpp synthesis. A second S. coelicolor RelA homologue (RshA, with 42% identity to S. coelicolor RelA) was identified in the genome sequence. However, deletion of rshA had no effect on the ability of the relA mutant to make Act and Red when grown under conditions of phosphate limitation. While high-level induction of tipAp::rshA in the relA mutant resulted in growth inhibition, low-level induction restored antibiotic production and sporulation. In neither case, nor in the relA mutant that was grown under phosphate limitation and producing Act and Red, could (p)ppGpp synthesis be detected. Thus, a ppGpp-independent mechanism exists to activate antibiotic production under conditions of phosphate limitation that can be mimicked by overexpression of rshA.     

Nonsense and insertion mutants in the relA gene of E. coli: cloning relA.

We have made use of lysogens of a specialized transducing bacteriophage, lambdapyrG+ relA+, to select nonsense (relAnon) and insertion (relAins) mutations in the relA gene. Three independent relAnon mutants were isolated on the phage. In all three, the relaxed phenotype was suppressed by supD, supE, supF or sup6. Three independent relAins mutants were isolated, all containing an insertion element (probably IS2) in an apparently identical location in the relA gene. Polyacrylamide gel electrophoretic analysis of peptides synthesized by the phages in ultraviolet lightkilled host cells revealed that no stringent factor was coded for by either the relAins or relAnon phages (the latter in a sup+ cell); stringent factor was detected when the relAnon phages were used in a similar experiment with supD or supE host cells. The relAnon and relAins mutations could be crossed in haploid form in the E. coli chromosome. These recombinants grew with a normal doubling time, had a ppGpp pool which was between 70 and 100% compared with the classical relA strain, and underwent a normal carbon source shift-down. A restriction endonuclease map of the pyrG relA region of the specialized transducing phage is presented in which the position of the insertion element (recognized by a novel Hind III-cut site) defines the position of the relA gene. This position was verified by an analysis of the structure of five plasmids formed by cloning portions of the region in the pBR322 cloning vehicle. Our results indicate that the relA gene is not an essential cellular function, that there might be a second mechanism for the synthesis of basal level ppGpp in the cell and that the sole function of the relA gene is apparently the high level ppGpp synthesis triggered in response to deacylated tRNA.     

Relaxed mutants of Escherichia coli RNA polymerase

When Escherichia coli cells are treated with either polymixin or gramicidin at concentrations that block protein and RNA synthesis, they accumulate a significant amount of guanosine tetraphosphate ppGpp. Such accumulation occurs in stringent (relA+) as well as in relaxed (relA) strains and no guanosine pentaphosphate pppGpp is then detected within the cells. These observations suggest that polypeptide antibiotics elicit ppGpp formation through a mechanism different from the stringent control system triggered by amino acid starvation of bacteria. Experiments based on tetracycline action indicate, moreover, that the accumulation of ppGpp under polymixin or gramicidin treatment is connected with a strong restriction of the degradation rate of this nucleotide.     

SpoT governs Legionella pneumophila differentiation in host macrophages

During its life cycle, Legionella pneumophila alternates between a replicative and a transmissive state. To determine their contributions to L. pneumophila differentiation, the two ppGpp synthetases, RelA and SpoT, were disrupted. Synthesis of ppGpp was required for transmission, as relA spoT mutants were killed during entry to and exit from macrophages. RelA, which senses amino acid starvation induced by serine hydroxamate, is dispensable in macrophages, as relA mutants spread efficiently. SpoT monitors fatty acid biosynthesis (FAB), since following cerulenin treatment, wild-type and relA strains expressed the flaA transmissive gene, but relA spoT mutants did not. As in Escherichia coli , the SpoT response to FAB perturbation likely required an interaction with acyl-carrier protein (ACP), as judged by the failure of the spoT-A413E allele to rescue transmissive trait expression of relA spoT bacteria. Furthermore, SpoT was essential for transmission between macrophages, since secondary infections by relA spoT mutants were restored by induction of spoT , but not relA . To resume replication, ppGpp must be degraded, as mutants lacking spoT hydrolase activity failed to convert from the transmissive to the replicative phase in either bacteriological medium or macrophages. Thus, L. pneumophila requires SpoT to monitor FAB and to alternate between replication and transmission in macrophages.     

Studies on the turnover of endogenous choline-containing phospholipids of cultured neuroblastoma cells

The effects of two polypeptide antibiotics, polymixin B and gramicidin S, on the intracellular pool size and turnover of guanosine tetraphosphate (ppGpp) were analyzed in stringent (relA+) and relaxed (relA) strains of Escherichia coli. When either one of these two drugs was added to stringent bacteria cultures at a final concentration that blocked protein and RNA synthesis, ppGpp was found to accumulate. Under similar conditions of inhibition of macromolecular synthesis, ppGpp also appeared to accumulate in relaxed bacteria. Moreover, in either type of strain, no significant accumulation of guanosine pentaphosphate (pppGpp) could be detected upon drug treatment. It was, therefore, concluded that polymixin and gramicidin elicit ppGpp accumulation through a mechanism independent of the relA gene product and, consequently, quite distinct from the stringent control system triggered by amino acid starvation. Further experiments performed by using tetracycline as an inhibitor of ppGpp synthesis, showed that the increase in the level of this nucleotide induced by drug action was due, in fact, to a strong restriction of its degradation rate.